Engineer Escherichia coli for 1-propanol production by integrating novel biotechnological and bioprocessing strategies

现代生物技术的进步大大扩展了大肠杆菌（E. coli）的能力，因此它现在是生物制造中最受欢迎的细胞工厂之一。具体来说，基于重组DNA技术的基因工程允许引入外源基因进行体外表达。基因组工程，基于位点特异性基因敲入和敲除技术，实现最佳的基因组编辑。代谢工程能够控制和调节关键的代谢通量，以过量产生目标代谢物。合成生物学允许人们移植外来途径来产生非天然的代谢物。提出的发现资助计划将寻求一种新的方法，将这些生物技术策略结合起来，实现大肠杆菌菌株的“基因剪裁”，用于工业目的的最终目标。1-丙醇是一种具有多种工业用途的精细化学品，在替代乙醇作为生物燃料方面具有很强的潜力。然而，到目前为止，1-丙醇的生产主要依靠化学合成，还没有发现微生物是天然的1-丙醇生产者。该野生型大肠杆菌1-丙醇非天然代谢物将作为技术示范的目标产品，所开发的策略可普遍应用于生产其他高价值代谢物。最近，我的研究小组通过操纵睡美人变异酶（Sbm）操纵子，在大肠杆菌中发现了一种新的1-丙醇生物合成方法。这个四基因操纵子（sbm-ygfD-ygfG-ygfH）编码参与钴胺依赖代谢途径的各种酶，将琥珀酸脱羧为丙酸。通过在Sbm操纵子内表达琥珀酸盐广泛异化的某些基因以及增加各种前体可用性的关键基因，成功地证明了工程大肠杆菌菌株的异源生产1-丙醇。为了提高1-丙醇的产量，建议的研究计划将针对基于生物技术和生物加工策略的新系统方法来解决各种问题，以优化该生物转化系统。与菌株构建相关的基本生物技术问题包括：(1)从各种微生物中寻找和鉴定参与1-丙醇生物合成的新基因；(2)大肠杆菌的代谢工程，将中心碳通量导向1-丙醇生产途径；(3)大肠杆菌的基因组工程，敲入和敲除影响1-丙醇生产的各种关键基因。另一方面，与栽培系统相关的应用生物处理问题包括：(1)培养条件的一般表征，如pH、温度、培养基配方、好氧或厌氧培养、可替代的廉价碳源；(2)批、补料批和恒化培养的操作方案和控制策略的开发；(3)在各种遗传和生物处理背景下的代谢通量的数学建模和分析，以确定限制1-丙醇生产的潜在步骤。提出的探索研究计划提供了一个独特的培训计划，在新的科学和工业生物技术。学员将获得未来生物制造职业所需的广泛的高级技能。除了对与生物制造相关的各种新型生物技术的广泛科学理解外，开发的1-丙醇生产的生物菌株和生物工艺可以很容易地转移到加拿大生物产业进行商业化，从而提高加拿大在生物制造和生物燃料方面的技术领先地位。

Advances in modern biotechnology have significantly extended the capacity of the bacterium Escherichia coli (E. coli) so that it is now one of the most popular cell factories for biomanufacturing. Specifically, genetic engineering based on recombinant DNA technology allows the introduction of foreign genes for episomal expression. Genomic engineering, based on site-specific gene knock-in and knock-out technology, enables optimum genomic editing. Metabolic engineering enables both control and tuning of key metabolic fluxes to overproduce target metabolites. Synthetic biology allows one to graft foreign pathways to produce non-natural metabolites. The proposed Discovery Grant program will pursue a novel approach that integrates these biotechnological strategies to realize the ultimate goal of “genetic tailoring” of E. coli strains for industrial purposes. 1-Propanol is a fine chemical with various industrial applications and has strong potential to replace ethanol as an alternative biofuel. However, up to now, 1-propanol production primarily relies on chemical synthesis and no microorganisms have been identified as a natural 1-propanol producer. This non-native metabolite of 1-propanol for wild-type E. coli will be used as the target product for technological demonstration and the developed strategies can be generically applied to produce other high-value metabolites. Recently, my research group identified a novel biosynthesis of 1-propanol in E. coli by manipulating the sleeping beauty mutase (Sbm) operon. This four-gene operon (sbm-ygfD-ygfG-ygfH) encodes various enzymes involved in a cobalamin-dependent metabolic pathway for decarboxylation of succinate into propionate. By expressing certain genes within the Sbm operon for extensive dissimilation of succinate along with key genes for increasing the availability of various precursors, heterologous production of 1-propanol in engineered E. coli strains was successfully demonstrated. To enhance 1-propanol production, the proposed research program will target a new systematic approach based on biotechnological and bioprocessing strategies to address various issues to optimize this biotransformation system. Fundamental biotechnological issues associated with strain construction include: (1) search and identification of novel genes involved in biosynthesis of 1-propanol from various microorganisms, (2) metabolic engineering of E. coli to drive the central carbon flux towards 1-propanol production pathway, (3) genomic engineering of E. coli to knock in and knock out various key genes affecting 1-propanol production on the genome. On the other hand, applied bioprocessing issues associated with the cultivation system include: (1) generic characterization of cultivation conditions, such as pH, temperature, medium recipe, aerobic or anaerobic cultivation, alternative cheap carbon sources, (2) development of operating protocols and control strategies for batch, fedbatch, and chemostat cultivations, (3) mathematical modeling and analysis of metabolic fluxes under various genetic and bioprocessing backgrounds to identify potential steps limiting 1-propanol production. The proposed Discovery research program provides a unique training program in novel scientific and industrial biotechnologies. Trainees will obtain a wide range of advanced skills required for future careers in biomanufacturing. In addition to extensive scientific understanding of various novel biotechnologies associated with biomanufacturing, the developed biological strains and bioprocess for 1-propanol production can be readily transferred to the Canadian bio-industry for commercialization, boosting Canada’s technological leadership in biomanufacturing and biofuels.